Eastern and Western equine encephalitis viruses (EEEV and WEEV; Alphavirus; Togaviridae) are mosquitoborne alphaviruses causing severe encephalitis in humans. There are no human vaccines for alphaviruses in case of widespread disease. We have previously developed mouse models describing pathogenesis of these alphaviruses and have shown that cationic-liposome-DNA complexes (CLDC) elicit protective activation of innate immunity in mice following WEEV challenge. In this proposal, CLDC-based vaccine platforms (LANAC) will be developed that include E2-E1 and E1 glycoproteins having novel N-glycans. We will test the hypothesis that immunization with WEEV E2-E1 or E1-based LANAC vaccines elicits protective immunity against multiple alphaviruses. The specific aims of the proposal are as follows: Aim 1) Construct and produce recombinant forms of the WEEV envelope glycoproteins, E1 and E2-E1 using a conventional baculovirus-insect cell system and a baculovirus-insect cell system with a humanized protein A/-glycosylation pathway. Aim 2) Evaluate protection provided by recombinant envelope WEEV glycoproteins (invertebratetype A/-glycans or vertebrate-type A/-glycans) as subunit vaccines in mice when used in combination with CLDC's. Protection will be evaluated following subcutaneous and mucosal immunization in the mouse model and aerosol-, subcutaneous-, and mosquito-delivered virus challenge. Aim 3) Identify the humoral and cellular immunological mechanisms responsible for the most efficient protection of mice following exposure to the WEEV E1 and E2-E1 glycoprotein vaccines with CLDC adjuvant. These immunological responses include quantifying IgG, IgM and IgA antibody and neutralizing antibody titers and mapping potentially protective T cell epitopes to WEEV glycoproteins. We will also compare immune responses to E1 and E2-E1 glycoproteins in immunized CD1, B6, mice which lack B-cells, and T-cell depleted mice. Aim 4) Characterize the ability of WEEV E1 glycoprotein to provide cross-protection in mice to other alphaviruses. Cross protective glycoproteins with CLDC's will be evaluated for EEEV, VEEV, and Sindbis virus (SINV) at CSU and Chikungunya and Ross River viruses (CHIKV and RRV) at UNC-Chapel Hill as part of a collaboration with Dr. Robert E. Johnston's lab and SERCEB. We will evaluate protection against multiple virus challenge routes and evaluate the longevity of immunological responses and protection. This research project fits within the RMRCE Integrated Research Focus on Immunomodulation, Adjuvants and Vaccines, and will interact with RP1.5, RP1.6, RP 3.3 and utilize the resources of Core C and E.